Method for isolation of highly pure von willebrand factor

ABSTRACT

The invention relates to a method for isolation of highly pure von Willebrand Factor in which recombinant von Willebrand Factor (rvWF) is chromatographically purified by anion exchange chromatography on an anion exchanger of the quaternary amino type in a buffer solution comprising buffer substances and optionally salt. 
     The buffer solutions are preferably free of stabilizers, amino acids and other additives. According to this method, highly pure recombinant vWF can be obtained, which is free from blood plasma proteins, especially free from Factor VIII, and is physiologically active. 
     Further, the invention relates to a pharmaceutical preparation that contains rvWF, which is comprised of multimers with a high structural integrity.

This application is a division of application Ser. No. 08/653,298, filedMay 24, 1996, which claims section 120 priority from PCT/EP95/03892,filed Oct. 2, 1995.

The invention relates to a method for the isolation of a highly pure vonWillebrand Factor (vWF). Further, the invention relates to recombinantWillebrand Factor (rvWF), which is obtainable according to the method ofthe invention as well as a pharmaceutical composition comprising rvWF.

In blood coagulation, the transition of liquid blood occurs in the bloodclot, a gelatinous mass which brings about the sealing of injured bloodvessels through thrombosis. Thereby, the transformation of solublefibrinogen present in plasma into the fibrous, gelatinous coagulationmaterial, fibrin, occurs in a multi-step process (so-called bloodcoagulation cascade) involving at least 15 different blood clottingfactors characterized with roman numerals, each of which, whenactivated, activates the respective next inactive step.

Of the coagulation factors, calcium ions, fibrinogen and prothrombin(Factor II) constantly circulate in blood, others are activated bytissue injury or contact with collagen or phospholipids fromthrombocytes (Factor XII). Numbered among the common blood-clottingfactors are several serine proteases such as kallikrein, thrombin andthe activated Factors VII, IX, X and XI.

The thrombocytes bind to collagen of the injured connective tissue inthe presence of von Willebrand Factor (a component of the coagulationFactor VIII) through adhesion. They alter their form and developpseudopods, and in addition to this, their outer membrane facilitatesadhesion to other thrombocytes. Thereafter, they release varioussubstances from their granula, whereby vasoconstriction as well as theaccumulation and activation of other factors of plasmatic coagulationare mediated.

During normal blood coagulation, direct and indirect functions areassigned to von Willebrand Factor. It binds in a complex to Factor VIII.This complex serves to stabilize Factor VIII. This stabilized FactorVIII has then essential cofactor functions in the activation of FactorX. However, von Willebrand Factor also directly influences bloodcoagulation since it mediates platelet aggregation to injured vessels.

In hemophilia, blood coagulation is impaired by a deficiency in certainplasmatic blood coagulation factors. In hemophilia A, the bleedingtendency is based on a deficiency in Factor VIII, and/or a deficiency invWF which constitutes an essential component of coagulation Factor VIIIcomplex. Hemophilia A can be treated by replacement of the missingcoagulation factor by factor concentrates from conserved blood, forexample by intravenous infusion of Factor VIII, a vWF/Factor VIIIcomplex or vWF.

There are many syndromes which can be traced back to under- oroverproduction of von Willebrand Factor. Hence, an overproduction of vWFleads, for example, to an increased tendency toward thrombosis, whereasa decrease in vWF results in an increased bleeding tendency or prolongedbleeding time.

Von Willebrand Syndrome can manifest itself in several forms. All formswhich may result from an absolute absence of functional vWF aredistinguished by a prolonged bleeding time. A deficiency in vWF can alsocause phenotypic hemophilia A because vWF is an essential component offunctional Factor VIII. In these cases, the half-life of Factor VIII isdecreased to such an extent that it can no longer perform its particularfunctions in blood-clotting.

vWF circulates in plasma in a concentration of 5-10 mg/l in the form ofa non-covalent complex with Factor VIII. vWF is a glycoprotein which isformed in various cells of the human body and is later released into thecirculation. Thereby, starting from a polypeptide chain with a molecularweight of approximately 220,000 (vWF monomer) in cells, vWF dimer(primary vWF dimer) is formed with a molecular weight of approximately550,000 by formation of several disulfide bridges. Then vWF dimers withincreasing molecular weights up to approximately 20 million are producedthrough coupling further polymers of vWF. It is suspected thatespecially the high molecular weight vWF fractions have an essentialimportance in blood coagulation.

Various methods for purifying and concentrating vWF are described in theliterature, all of which use human blood plasma as a source for the vonWillebrand Factor.

Structural analysis of vWF can be undertaken with high resolutionelectrophoretic methods. In this way, it was found by Baillod et al.,Thrombosis Research 66, 745-755, 1992, that vWF multimers are separatedinto bands and each multimer band carries one or several satellite bandswith it. This appearance is traceable back to the proteolyticdegradation of vWF multimers. The simple addition of protease inhibitorsto blood samples could not inhibit this degradation.

Abnormal vWF of type IIA demonstrates an altered electrophoresispattern. As a result of the multimer analysis, it was found that inpatients with von Willebrand disease of type IIA, the multimers eachappear as single bands (singlets) and are not cleaved in satellitebands. This is traceable back to the fact that a protease-sensitive bondbetween Tyr-842 and Met-843 in the type IIA patients is possibly notcleaved. These patients demonstrate different syndromes in connectionwith a bleeding tendency.

A similar picture of the multimer bands for recombinant vWF is describedby Fischer et al., FEBS Letters 351 (1994) 345-348. This rvWF isexpressed in CHO cells and a multimer analysis was undertaken. Incontrast to plasma vWF, no triplet structure was observed. Consequently,this rvWF is present as completely intact protein which is notproteolytically degraded.

However, the rvWF was not subject to any treatment methods such as apurification, virus inactivation and/or virus depletion. Consequently apharmaceutical preparation was still not present.

The vWF preparations described in the prior art comprise vWF in aproteolytically degraded form. The stability of these preparations isthereby limited. Also, experiments to prevent proteolysis after taking ablood sample with suitable inhibitors did not lead to vWF with intactstructure.

EP-A-0 503 991 describes the purification of vWF from humancryoprecipitate by three successive chromatography steps: 1.ion-exchange chromatography on DEAE (DEAE cellulose, diethylaminoethylcellulose) Fractogel and elution of vWF by 0.15M NaCl; 2. furtherion-exchange chromatography on DEAE-Fractogel and elution of the vWF by0.17M NaCl; and 3. affinity chromatography on gelatin Sepharose®.Buffers which contained amino acids and calcium ions were used as buffersystems.

M. Burnouf-Radosevich and T. Burnouf, Vox Sang 62 (1992) 1-11 describe asimilar chromatographic purification of plasma vWF by a combination ofion-exchange chromatography on DEAE-Fractogel with a gelatin Sepharose®filtration in a buffer system containing amino acids and calcium ions.

WO-A-8 912 065 describes the separation of plasma proteins from plasmacryoprecipitates through binding of the proteins on DEAE-Fractogel andthrough step-wise elution by increasing addition of NaCl. The method issuitable especially for isolation of Factor VIII concentrate of highpurity for treating hemophilia A, as well as for isolation ofconcentrates of fibrinogen, vWF and fibronectin. The fractionscontaining vWF are preferably subjected to a second chromatography onthe same anion exchanger using a buffer containing amino acids andcalcium ions.

EP-A-0 416 983 describes the isolation of a vWF-Factor VIII complex fromhuman plasma by precipitation with a combination of barium chloride andaluminum hydroxide, and subsequent anion exchange chromatography onDEAE-Fractogel.

According to Harrison et al., Thrombosis Research 50 (1988) 295-304,vWF/Factor VIII complex is purified by chromatography on dextranesulfate agarose.

However, in the purification of vWF-Factor VIII complex according tothese methods, Factor VIII:C should be obtained in higher purity.

Therefore, in the treatment of hemophilia A, continuously betterpurified Factor VIII:C concentrates are employed which do not containvWF or only contain vWF in trace amounts. Therefore, such preparationsare not suitable for the treatment of vWF deficiency. The need for purevon Willebrand Factor concentrate is therefore very great.

EP-A-0 469 985 and U.S. Pat. No. 5,252,710 describe a method forproduction of vWF from plasmacryoprecipitate which is extensively freefrom Factor VIII, in which vWF is separated from Factor VIII in a firstpurification step because vWF is not bound to the anion exchange column,but rather only to Factor VIII. Then, in a second step, the saltconcentration of the material not bound to the anion exchanger issubstantially decreased and vWF is bound to a second anion exchanger andthen further eluted with a solution of higher ionic concentration.

DE-A-3 904 354 describes the production of a vWF concentrate from plasmacryoprecipitate by separation of vWF from Factor VIII, whereby FactorVIII, but not vWF, is bound to an ion exchanger.

U.S. Pat. No. 5,252,709 describes a method for the separation of FactorVIII, vWF, fibronectin and fibrinogen from human plasma, whereby FactorVIII, vWF and fibronectin are first bound to an ion exchanger of theDEAE type and subsequently separately eluted with increasing saltconcentration from the ion exchanger.

Although these methods describe the purification of vWF with separationof Factor VIII, a low level of contamination with Factor VIII and/orwith other blood plasma proteins cannot be excluded.

Additionally, all vWF concentrates which are obtained by isolation ofthe protein from human blood plasma or come in contact with biologicalmaterial from mammals are potentially at a risk of containing pathogenicmolecules from plasma donors such as, for example, viruses.

The object of the present invention relates to making available anefficient, easy and safe method for the production of highly pure vonWillebrand Factor which is essentially free from other plasma proteinsand especially free from Factor VIII.

A further object of the invention is to make a pharmaceutical available,which comprises a vWF whose stability is improved in comparison topreviously known preparations.

This object is solved with the subject-matter of the present invention.

Subject-matter of the present invention is a method for the isolation ofhighly pure von Willebrand Factor in which recombinant von WillebrandFactor (rvWF) is purified by anion exchange chromatography on an anionexchanger of the quaternary amino type.

Preferably, the rvWF purified by anion exchange chromatography isfurther purified by affinity chromatography on immobilized heparin in abuffer solution comprising buffer substances and optionally salt.

Recombinant von Willebrand Factor is isolated from the cell-free culturefiltrate of transformed, virus-free, animal cells by means of cellculture techniques.

Preferable embodiments include methods where the rvWF in the form of aconcentrate is purified from cell-free culture supernatants oftransformed cells. The buffer system for use with the invention ispreferably free of stabilizers, amino acids and other additives.

Preferably, the anion exchange chromatography and/or affinitychromatography used according to the invention is carried out at a pHrange of 6.0-8.5, and more preferably at a pH value of 7.4. The rvWFbound in on the anion exchanger or in affinity chromatography onimmobilized heparin is preferably eluted by increasing the saltconcentration. Preferably, the quaternary anion exchanger for useaccording to the invention is a Fractogel with tentacle structure, suchas EMD-TMAE Fractogel.

Preferably, the rvWF is bound to the anion exchanger at a saltconcentration <270 mM, and eluted at a salt concentration >270 mM andpreferably at >280 mM. The anion exchange chromatography can be carriedout on a carrier with heparin bound thereon, wherebyAF-Heparin-Toyopearl®, Heparin EMD-Fractogel® or Heparin Sepharose FastFlow® can be employed.

The rvWF pre-purified in anion exchange chromatography can be bound toimmobilized heparin at a salt concentration <150 mM and is eluted at asalt concentration >150 mM, preferably at 200-300 mM, more preferably160-270 mM.

The salt that can be employed according to the invention is preferablymonovalent and divalent. A preferred salt is NaCl.

Further subject-matter of the present invention is a recombinant vonWillebrand Factor which is free from blood plasma proteins, especiallyfree from Factor VIII, and is obtainable according to the method of theinvention. This recombinant von Willebrand Factor is physiologicallyactive.

Further subject-matter of the present invention is the use of rvWF, inparticular the rvWF obtainable according to the method of the invention,for treatment of hemophilia A, hemophilia A with a deficiency in vWF andvarious forms of von Willebrand disease. Further subject-matter is alsothe use of recombinant von Willebrand Factor for the production of apharmaceutical composition for treating hemophilia A and hemophilia Awith a deficiency in vWF. Further subject-matter is the use of rvWFobtainable according to the method of the invention for the treatment ofvarious forms of von Willebrand's disease. Further subject-matter of thepresent invention is a pharmaceutical composition characterized in thatit comprises nonfragmented rvWF obtainable according to the method ofthe invention in a physiologically acceptable carrier.

Further subject-matter is a stable preparation comprising virus-safervWF, which comprises multimers with high structural integrity. Themultimers can be derived from a rvWF containing fraction obtainable by achromatographic purification method, whereby the multimers are notproteolytically degraded. The rvWF can have multimer bands in theabsence of satellite bands after electrophoresis analysis. ThervWF-containing fraction can obtained from a cell culture.

The preparation can be formulated to a pharmaceutical preparation foradministration to patients without causing side-effects such as theformation of thrombi, thrombocyte activation or thrombocytopenia.Preferably, the preparation can remain stable in solution at roomtemperature for at least 50 hours. More preferably, the preparation isin a form acceptable for infusion.

The preparation can comprise salts, such as sodium chloride and calciumchloride, and amino acids such as glycine and lysine, preferably in a pHin the range of 6-8. Preferably, the preparation according is treatedfor inactivation and/or depletion of viruses. The pharmaceuticalpreparation comprising rvWF can have a multimer pattern with a singletstructure that is maintained after administration to a mammal.

The invention also pertains to a method for the treatment of hemophiliaA, which comprises the steps of administering to a patient in atherapeutically effective amount of rvWF. This method also can be usedto treat von Willebrand Factor deficiency.

The vWF preparation according to the invention can be added topreparations containing Factor VIII, recombinant Factor VIII orfunctional deletion mutants of Factor VIII.

Recombinant vWF (rvWF) is isolated from cell-free culture medium afterfermentation of animal cells and purified. The culture medium used forfermentation constitutes a complex, synthetic mixture of all materialsfor maintaining animal cells which are customary for this purpose suchas vitamins, sugars, salts, hormones, antibiotics and buffer substances,and therefore, is essentially different in all fundamental propertiesfrom the composition of human blood plasma or plasma cryoprecipitate. Itwas not to be predicted therefore that the method according to theinvention would be outstandingly suitable for the production of highlypure von Willebrand Factor. Preferably, a recombinant vWF concentratefrom cell-free culture supernatants of transformed cells is employed inthe method according to the invention.

In the method according to the invention a buffer solution is preferablyused for a buffer system which is comprised of buffer substances whichare preferably free from stabilizers, amino acids and other additivesand optionally salt, preferably sodium chloride. It is known from theprior art that stabilizers, amino acids and other additives arenecessary in order to, on the one hand stabilize von Willebrand Factorand, on the other hand, to destabilize the Factor VIII-von Willebrandcomplex and to ease the separation of other proteins. In the methodaccording to the invention, use of such components in the buffer can beentirely refrained from and despite this, a physiologically activerecombinant von Willebrand Factor is obtained.

A buffer system free from stabilizers, amino acids and other additivesis preferably used as a buffer system, such as for example,Tris-HCl/NaCl buffer, phosphate buffer and citrate buffer.

Anion exchange chromatography and/or affinity chromatography arepreferably carried out in a pH range of 6.0-8.5 and particularlypreferably at a pH value of 7.4.

The elution of rvWF bound to the anion exchanger in anion exchangechromatography and bound to immobilized heparin in affinitychromatography preferably occurs by increasing the salt concentration.

Fractogel® with tentacle structure, and preferably EMD-TMAE Fractogel®is used as an anion exchanger of the quaternary amino type.

Preferably, rvWF is bound to the anion exchanger at a salt concentrationof <270 mM, and eluted at a salt concentration >270 mM, and preferablyat >280 mM. Soluble monovalent and divalent salts are usable as salts,whereby NaCl is preferred.

Any carrier to which heparin is bound can be used for affinitychromatography. For example, AF Heparin Toyopearl® (a synthetic largepore, hydrophilic polymer based on methacrylate; Tosohaas), HeparinEMD-Fractogel® (a synthetic hydrophilic polymer based on ethyleneglycol,methacrylate and dimethacrylate; Merck) or Heparin Sepharose Fast Flows®(containing natural dextran and/or agarose derivatives; Pharmacia) haveproven themselves to be well-suited.

Preferably, the rvWF pre-purified by the step of anion exchangechromatography is bound to immobilized heparin at a salt concentrationof <150 mM and eluted at a salt concentration of >150 mM, preferablyfrom 200-300 mM and more preferably 160 MM to 270 mM. Monovalent anddivalent salts are usable as salts, whereby NaCl is preferred.

Based on the molecular weight of rvWF (molecular weight of 500,000 toseveral million) such carrier materials are preferably used in themethod according to the invention in anion exchange chromatography aswell as in affinity chromatography which do not impede the rvWF moleculein its diffusion and distribution within the carrier structure, such as,for example, gels with tentacle structure.

In a preferred embodiment of the method according to the invention, thecell-free culture medium is first filtered on a strong anion exchanger,whereby the rvWF is bound by the exchanger. A large pore gel withtentacle structure and with strong binding ion exchange groups of thequaternary amino type, such as for example EMD-TMAE-Fractogel® ispreferably used as an ion exchanger. After removal of the accompanyingproteins and impurities by means of salt-containing buffer, preferablyNaCl-containing buffer, rvWF is then eluted from the ion exchanger inenriched form. In the second purification step of affinitychromatography, the eluate containing rvWF is brought into contact withan affinity carrier with covalently bound heparin, whereby rvWF binds tothis carrier. After the removal of foreign substances and foreignproteins by a suitable elution substance (such as, for example, buffersubstance), rvWF is eluted from the affinity carrier, preferably bymeans of a NaCl-containing buffer system.

A highly pure rvWF can be obtained according to the method of theinvention for isolation of a highly pure von Willebrand Factor, which isfree from antibodies, free from blood plasma proteins and especiallyfree from Factor VIII, which is physiologically active and which is freefrom pathogenic viruses.

The highly pure rvWF is further characterized in that the portion of vWFprotein to total protein is at least 80%, especially at least 86%.

The highly pure rvWF obtainable according to the method of the inventioncan be employed in a targeted manner in the treatment of hemophilia A orhemophilia A with a deficiency in vWF as a result of its properties:that it is free from antibodies, free from plasma proteins, free frompathogenic viruses and free from Factor VIII.

Furthermore, the highly pure rvWF obtainable according to the method ofthe invention can be used for treatment of various forms of vonWillebrand disease.

Further, according to the invention, a stable preparation is madeavailable, which comprises rvWF consisting of multimers with a highstructural integrity. This stable preparation is preferably formulatedto a pharmaceutical preparation. The rvWF is so stable that it can bemade available as a virus-safe preparation. The virus safety isguaranteed by method steps for treating the rvWF for inactivation ofviruses and/or depletion of viruses.

A heat treatment in solution and/or in the solid state which canreliably inactivate lipid coated as well as non-lipid coated viruses isespecially suited for the inactivation of viruses. For example, thepreparation according to the invention is heat treated in a solid, wetcondition according to EP-0 159 311. Other methods for virusinactivation also encompass a treatment with detergent or chaotropicsubstances, for example according to EP 0 519 901, WO 94/13329, DE 44 34538, EP 0 131 740 and WO 90/15613.

rvWF is preferably contained in the preparation according to the presentinvention as a highly pure protein which is obtained by achromatographic purification method. The chromatographic purificationparticularly occurs by ion exchange chromatography and/or affinitychromatography. For this, materials for anion exchange can be enlisted,among them synthetic carrier materials or carriers based oncarbohydrates with ligands, such as DEAE, TMAE, QAE, Q or aminoalkylgroups and/or carriers with immobilized substances which have a specificaffinity for vWF. Suitable affinity materials contain heparin forexample. This purification method is suitable for large-scale isolationof rvWF.

Additionally, it is to be noted that the rvWF in the preparationaccording to the invention has a surprisingly sufficient resistanceagainst proteolytic degradation such that the addition of customarystabilizers can be dispensed with. However, in exceptional cases, asuitable protease inhibitor can also be added during the productionmethod in order to obtain the intact structure. For further support ofthe stability of vWF, the stable preparation, in particular thepharmaceutical preparation, can also comprise a polyalkyleneglycol suchas PEG or polypropyleneglycol or glycerin in a concentration which doesnot precipitate rvWF and is physiologically acceptable.

According to the invention, the rvWF in the preparation has multimerbands in the absence of satellite bands after electrophoretic analysis.This corresponds to the structure of vWF, i.e. a non-fragmented, intactprotein. Preferably, rvWF in the stable or pharmaceutical preparationhas the entire spectrum of multimers, which is similar to nativemultimer distribution, especially the vWF with high molecular weight.

The stability of the preparation according to the invention is necessaryabove all for a liquid preparation. A solution of the preparationaccording to the invention is stable at room temperature, for examplefor at least 48 hrs., preferably for at least 72 hrs., and is storableat a temperature of 4° C. for more than 2 years. The stability is shownby an insignificant loss of activity of less than 50%, preferably lessthan 20%, and most preferably less than 10%, which is not substantial.Therewith, the preparation according to the invention is suitable as aninfusion preparation, which can also be infused into a patient over aperiod of several hours without risking changing the preparation ornecessitating a change in the dosage regime. With respect to theprevention of possible side-effect reactions, it is also advantageous toadminister a protein with intact and stable structure.

It has emerged that the pharmaceutical preparation according to theinvention can be administered to a patient without side-effect reactionssuch as formation of thrombi or microthrombi, thrombosis, thrombocyteactivation or thrombocytopenia. This was surprising above all becauservWF in the preparation according to the invention has a similarmultimer pattern to the form responsible for type IIA von Willebranddisease.

The formulation of the pharmaceutical preparation according to theinvention can occur in a known and customary manner, for example, withthe aid of salts and optionally amino acids, but it can also beperformed in the presence of tensides. Preferably, salts such as, forexample, sodium chloride or calcium chloride are used and a pH in therange of 6-8 is selected. As amino acids, glycine or lysine arepreferred. Equally, a pharmaceutically suitable buffer can be chosen. Asa result of the high stability of rvWF, the use of stabilizers, such ascarrier proteins or inhibitors, can usually be avoided.

The preferred concentration of rvWF in the administration-ready solutionis in the range of 1 to 100 units/ml. Because of the high purity of thepreparation, this can also be formulated in concentrations of up to 1000U/ml. The activity is characterized by ristocetin-mediated plateletaggregation and is given as ristocetin-cofactor activity (RCoF) (seeJournal of Clinical Investigations 52, 2708-2716, 1973 for this). Thenormal dose for vWF lies in the range of 40-80 RCoF units/kg inintervals of 6-48 hours. As an initial dose, a higher dose of up to 200RCoF can also be chosen.

With a biological half-life of more than 20 hrs., the half-life of rvWFafter administration of the preparation according to the invention issurprisingly clearly longer than for the preparations of the prior art.

According to a further aspect of the invention, the pharmaceuticalpreparation comprises rvWF which maintains the multimer pattern with asinglet structure even after administration to a mammal. Therewith, aproteolytic cleavage of the singlets to satellite bands is absent.

Preferably, the stable or pharmaceutical preparation according to theinvention comprises rvWF as a single essential ingredient. Therewith,this preparation can essentially comprise highly purified rvWF.

The rvWF obtainable according to the method of the invention can also beused for a stabilization of Factor VIII, of recombinantly producedFactor VIII or of functional deletion mutants of Factor VIII, wherebystabilization can be detected in vitro.

A Factor VIII preparation stabilized in this manner is not at risk, asare plasma products, of being contaminated with pathogenic viruses.

It was surprisingly found that rvWF possesses a potentially higherbinding capacity for Factor VIII, and therewith, binds Factor VIII moreefficiently than plasmatic vWF.

Therefore, subject-matter of the present invention is also a rvWFobtainable according to the method of the invention, characterized inthat it possesses increased binding capacity for Factor VIII.

For production of the stable or pharmaceutical preparations, the highlypure recombinant von Willebrand Factor-containing fractions arepreferably concentrated, and the concentrate is then further processed.

The pharmaceutical compositions can be present in a form for treatmentof hemophilia A, hemophilia A with deficiency in vWF and in variousforms customary and usual for administration in von Willebrand disease;preferably, they are present in a form of a preparation suitable forinfusion. In the following Examples, the invention is more closelyillustrated without limiting it to them.

Example 1 describes the purification of rvWF from cell-free culturemedium after fermentation of transformed animal cells by anion exchangechromatography. A continuing purification by the processing step ofaffinity chromatography is described in Example 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an 8% SDS-page separation of rvWF. Lane A: culturemedium; Lane B: fraction 280 mM NaCl after Fractogel; Lane C: 270 mMNaCl fraction after heparin affinity chromatography; Lane D: molecularweight marker.

EXAMPLE 1

Purification of rvWF from Culture Supernatants by Anion ExchangeChromatography

Recombinant vWF was isolated according to customary methods afterinfection of Vero cells (monkey kidney cells) with vaccinia virus incell culture. Vero/vaccinia expression systems and cell cultureconditions are described in detail in F. G. Falkner et al., Thrombosisand Haemostasis 68 (1992) 119-124; N. Barret et al., AIDS Res. 5 (1989)159-171 and F. Donner et al., AIDS Vaccine Research and Clinical Trials,Marcel Dekker, Inc, New York (1990). The expression of rvWF occurred ina synthetic DMEM standard medium (Dulbeccols minimal essential medium).

Recombinant vWF can also be isolated by transformation of CHO cells.

After fermentation of the transformed cells, the culture medium wasseparated and cells and cell fragments were removed by centrifugation.Further, smaller components, such as membrane fragments or bacteria wereremoved by filtration through a filter with a pore size of 0.4 μm.

770 ml cell-free culture supernatant was filtered with a flow rate of 2ml/cm² /min over a column (1.6 cm×5 cm, filled with 10 ml anionexchanger EMD-TMAE-Fractogel® (Merck)). The gel was previouslyequilibrated with 20 mM Tris-HCl buffer (pH 7.4). Subsequently, thecolumn was washed with 20 mM Tris-HCl buffer (pH 7.4).

Foreign materials were removed by washing the column with buffercontaining 200 mM NaCl. The rvWF was then eluted from the carrier with280 mM NaCl in 20 mM Tris-HCl buffer (pH 7.4). Subsequently, residualmaterial, which was possibly present, was eluted from the column with 1MNaCl. During chromatography, protein absorption was followed in acustomary manner at 280 nm. After chromatography, the proteinconcentration was determined according to the Bradford method (M.Bradford, Anal. Biochem. 72 (1976) 248-254). The content of rvWF wasdetermined by means of a commercial ELISA system (Boehringer Mannheim).

It was found that nearly the entire rvWF was bound to the carrier. rvWFwas eluted from the anion exchanger by 0.28M NaCl. The results of thepurification of rvWF on the anion exchanger are summarized in Table 1.

rvWF was enriched by 6-fold through the purification described in thisExample.

                  TABLE 1    ______________________________________                       Total               Volume  Protein   rvWF  rvWF/Total    Sample     (ml)    (μm/ml)                                 (μm/ml)                                       Protein    ______________________________________    Cell-Free  770     113       7.9   0.069    Supernatant    Elution    95      147       0.0016                                       0.00001    with 200 mM    NaCl    Elution    75      168       61    0.36    with 280 mM    NaCl    Elution    50      196       6     0.03    with 1 M    NaCl    ______________________________________

EXAMPLE 2

Purification of rvWF by Affinity Chromatography

rvWF obtained according to Example 1 was diluted with 20 mM Tris-HClbuffer (pH 7.4) to decrease the salt concentration (160 mM NaCl) .Subsequently, the solution was filtered through a column (1.6 cm×5 cm,filled with 10 ml AF heparin Toyopearl® 650 (Tosohaas)) with a flow rateof 1 ml/cm² / min. The column was previously equilibrated with 20 mMTris-HCl buffer (pH 7.4). Non-specifically bound proteins were firstremoved by washing with 20 mM Tris-HCl buffer (pH 7.4). rvWF was elutedfrom the carrier by 270 mM NaCl in 20 mM Tris-HCl buffer (pH 7.4).Finally, residual material was washed from the column with 1M NaCl.During chromatography, protein absorption was followed in a customarymanner at 280 nm. After chromatography, the protein concentration wasdetermined by means of the Bradford method (M. Bradford, l.c.). Thecontent of rvWF was determined by means of a commercial ELISA system(Boehringer Mannheim).

It was found that nearly the entire rvWF was bound to the column. Dugingelution with 270 mM NaCl, the large part of rvWF was eluted from thecolumn, whereas the washing with 1M NaCl contained only traces of rvWF.The results of this purification step are summarized in Table 2. Theportion of rvWF protein to total protein was increased to over 86% bythis purification step.

The fraction from 270 mM NaCl was more closely examined with denaturingSDS-protein gel electrophoresis (U. K. Laemmli, Nature 227, (1970)680-685) and subsequently with a Western-Blot.

As represented in FIG. 1, the denaturing electrophoretic analysisresulted in the fact that rvWF was isolated in high purity by thepurification described in Examples 1 and 2. In the product isolated inthis manner, no other coagulation factors, such as for example, FactorVIII, could be detected.

                  TABLE 2    ______________________________________                       Total               Volume  Protein   rvWF  rvWF/Total    Sample     (ml)    (μm/ml)                                 (μm/ml)                                       Protein    ______________________________________    rvWF       225     50          13.9                                       0.27    Concentrate    Elution    43      70        60    0.86    with 270 mM    NaCl    Elution    32      25         2    0.08    with 1 M    NaCl    ______________________________________

The purified rvWF possesses an activity of 4.32 U/mg rvWF:Ag withrespect to platelet aggregation.

EXAMPLE 3

Plasmatic vWF (p-vWF), vWF from cryoprecipitate (k-vWF) as well asrecombinant vWF (r-vWF) were purified by means of heparin affinitychromatography. The different vWF preparations were examined for theirbinding to Factor VIII.

                  TABLE 3    ______________________________________                Stochiometry    Sample      vWF:Factor VIII    ______________________________________    rvWF        2.0:1    k-vWF       2.6:1    p-vWF       3.0:1    ______________________________________

Table 3 shows the data of the stoichiometry of vWF: Factor VIII. Thedata shows that r-vWF possesses an essentially Ad higher bindingcapacity for Factor VIII than p-vWF.

EXAMPLE 4

Stability of recombinant von Willebrand Factor in Solution

A von Willebrand Factor preparation was prepared as described in Example2, and formulated in a buffer containing 5 g/l Na₃ citrate.2H₂ O, 2 g/lNaCl, 5 g/l glycine, 5 g/l L-lysine.HCl and 0.62 g/l CaCl₂. 2H₂ O, pH7.0, in such a manner that the von Willebrand concentration was 10 U/mlmeasured by means of ristocetin mediated platelet aggregation. Asolution of this type was held at 4° C., 25° C., 37° C. and 50° C. up to70 hours. At various times, samples were taken and measured for theirvon Willebrand Factor activity by means of the ristocetin mediatedplatelet aggregation.

At 4° C. and 25° C., no change in the activity was seen in theobservation time period, at 37° C. the activity remained over 80% forover 24 hours, and even at 50° C. no change in the biological activitycould be established over 8 hours. Simultaneously, the antigen contentwas ascertained by means of ELISA. The antigen content remained the sameas the starting value at all storage temperatures over the entiremeasurement period. The stability experiment was carried out without thecustomary protein stabilizers such as carrier proteins or sugar.

EXAMPLE 5

Lyophilization Behavior of Recombinant von Willebrand Factor

A recombinant von Willebrand Factor was formulated as described inExample 4, and adjusted to an activity of 10 U/ml. Then this wasdeep-frozen without further addition of common stabilizing agents andthen subsequently reconstituted to the starting volume with water.Thereafter, the ristocetin cofactor activity was newly determined. rvWFcould be reconstituted with a yield of 80%. As a comparative experiment,this was lyophilized in the presence of 0.1% human serum albumin;thereby 98% of the starting activity could be retained afterreconstitution.

EXAMPLE 6

Pharmacokinetics of Multimers of Recombinant von Willebrand Factor inPig

Von Willebrand deficient animals, such as for example the homozygous vonWillebrand deficient pigs described by Roussi et al., Brit J. Haematol.90: 661-668 1995, were used for the experiment. In this experiment afour month old female homozygous von Willebrand deficient pig weighing37 kg was employed. This was characterized by a bleeding time of over 30minutes measured according to the ear bleeding method of Samama et al.,Thromb. Haemostas. 71: 663-669, 1994, and a von Willebrand Factor plasmalevel under the detection limit was determined in antigen ELISA and bythe ristocetin cofactor activity. Factor VIII activity was approximately1 U/ml measured as human Factor VIII in the 1-step clotting test, 2-stepclotting test or chromogenic Factor VIII test (Immunochrom® FactorVIII:C, Immuno).

A preparation according to the invention which was isolated as describedin Example 2 was injected into the pig under anesthesia at a dose of 34RCoF U/kg body weight. Blood samples were taken at 30 min. 1 hr., 2hrs., 3 hrs., 6 hrs., 9 hrs., 12 hrs., 24 hrs., 32 hrs., and 48 hrs.after infusion and a citrate of plasma was produced from these.

From the plasma samples, the structure of von Willebrand Factormultimers was determined by SDS-agarose gel electrophoresis in a 2%agarose gel according to the method of Ruggeri et al., Blood 57:1140-1143. Thereby, the von Willebrand Factor multimers were madevisible by immune enzymatic staining according to Aihara et al., Thromb.Haemostas. 55: 263-267 1986. As a primary antibody, a rabbit-anti-vonWillebrand Factor-antiserum (Dakopatts, Glostrup, Denmark) was used at adilution of 1 5,000. An alkaline phosphatase conjugated affinitypurified goat-anti-rabbit-IgG H+L antibody (Axell, Accurate Chemical andScientific Corp., New York) in a dilution of 1:1,000 served as asecondary antibody. The staining of the protein bands occurred by meansof the nitroblue-tetrazolium-chloride-bromo-indolyl-phosphate substratesystem.

No von Willebrand Factor could be detected in the pig before thetreatment with the preparation according to the invention. Afteradministration of the preparation, a structure of a multimer patterncomprised of singlets atypical for the native condition, which wastraceable to a non-proteolytic digestion of von Willebrand Factor wasdemonstrated. This structural property remained unchanged over theentire observation time period, i.e. no proteolytic degradation of thepreparation occurred. Commensurate with the pharmacokinetics, thepreparation was successfully eliminated from the circulation. Multimersof the lowest molecular weight remained detectable up to 48 hours afterinfusion of the preparation.

A half-life of von Willebrand Factor according to the invention ofapproximately 30 hours could be calculated from the infusionexperiments. As a macroscopic parameter for the normalization of thecoagulation system disturbed in the deficient animal, the bleeding timewas determined, which could be corrected from over 30 minutes before theinfusion of von Willebrand Factor to approximately 13 minutes afterinfusion, whereby this effect was still detectable 32 hours after theinfusion.

We claim:
 1. A method for stabilizing Factor VIII, comprising contactingpreparations containing Factor VIII, recombinant Factor VIII orfunctional deletion mutants of Factor VIII with recombinant vonWillebrand Factor (rvWF) obtainable by anion exchange chromatography,wherein the recombinant von Willebrand Factor is in the form ofmultimers having high structural integrity and is not proteolyticallydegraded.
 2. The method for stabilizing according to claim 1, whereinthe recombinant von Willebrand Factor is obtained by:(a) contacting arvWF-containing composition with a quaternary amino type anionexchanger, wherein the contacting is undertaken using buffer having asalt concentration less than 270 mM; and (b) eluting the rvWF byincreasing the salt concentration above 270 mM, wherein the rvWF is inthe form of multimers having high structural integrity and is notproteolytically degraded.
 3. The method of stabilizing according toclaim 2, wherein the rvWF multimers have a singlet structure and lacksatellite bands.
 4. The method of stabilizing according to claim 2,wherein the buffer lacks (i) stabilizers and (ii) free amino acids.
 5. Amethod for stabilizing Factor VIII, comprising contacting preparationscontaining Factor VIII, recombinant Factor VIII or functional deletionmutants of Factor VIII with recombinant von Willebrand Factor (rvWF)obtained by:(a) contacting a rvWF-containing composition with aquaternary amino type anion exchanger, wherein the contacting isundertaken using buffer having a salt concentration less than 270 mM;and (b) eluting the rvWF by increasing the salt concentration above 270EM, wherein the rvWF is in the form of multimers having high structuralintegrity and is not proteolytically degraded able by anion exchangechromatography.
 6. The method of stabilizing according to claim 5,wherein the rvWF multimers have a singlet structure and lack satellitebands.
 7. The method of stabilizing according to claim 5, wherein thebuffer lacks (i) stabilizers and (ii) free amino acids.